The substantial costs and time involved in DNA sequencing are of concern, particularly in view of the "Human Genome Project", a research project that has the ultimate goal of sequencing the entire human genome. The currently preferred method of DNA sequencing is the "Sanger" (a.k.a. "dideoxy") sequencing method. See e.g. U.S. Pat. No. 4,795,699. See also "Sequenase Version 2, Step-By-Step Protocols for DNA Sequencing With Sequenase Version 2.0" by U.S. Biochemical (5th edition 1990). The disclosure of all patents and articles described herein are incorporated by reference as fully set forth herein.
In the dideoxy method, one inserts the DNA fragment to be sequenced into a circular phage template, then purifies the modified template, anneals a primer to the template, and generates therefrom four populations of single stranded DNA fragments that have one defined end, one variable end, and a radioactive portion. The variable end is a specific nucleotide (G, C, T, or A) in each grouping. The fragments are separated on the basis of their length on a high resolution gel. Each band corresponds to a specific nucleotide in the sequence, and this information is used to identify the position of the nucleotide in the sequence.
Some of the steps of this sequencing procedure have already been automated (e.g. DNA synthesis and gel analysis). However, purification of single stranded circular DNA has resisted efficient automation. One company has recently attempted to partially automate this step by developing a hybridization capture method. See G. Fry et al. 124 BioTechniques 124-131 (1992). Their method hybridizes a single stranded oligonucleotide to a complementary strand of a single stranded circular template. The oligonucleotide has an attached biotin group that is capable of binding to streptavidin-coated paramagnetic styrene beads. One end of the oligonucleotide hybridizes to the template and the other end binds (via the biotin group) to a magnetic anchor. A magnetic field is used to drag the beads (and thus the template) out of solution.
Unfortunately, this system suffers from the relatively weak hybridization of the oligonucleotide to the template. Using much longer oligonucleotides might lead to better hybridization, but would also cause other problems (e.g. cost of oligonucleotides).
In other unrelated work, there have been reports of some circular DNA that can surround a short DNA sequence on two sides to form a triple helix (E. Kool, 113 J. Am. Chem. Soc. 6265-6266 (1991)), and other reports that certain single stranded oligonucleotides can hairpin around both sides of a short non-circular single stranded oligonucleotide to form a triple helix (L. Xodo et al., 18 Nuc. Acids Res. 3557-3564 (1990)).